Project Summary/Abstract B cells are central to the pathogenesis of multiple sclerosis (MS), including B cell functions unrelated to production of immunoglobulins (Ig). Injury to gray matter (GM), particularly subpial GM, is a pathologic substrate of the progressive stages of MS, with cognitive and physical worsening experienced by many patients. Our hypothesis is that B cells from the peripheral immune system enter the spinal fluid and meninges, and release non-immunoglobulin (Ig) factors that injure oligodendroglia (OL) and neurons/axons in underlying cerebral cortical gray matter (GM) and slowly evolving perivascular lesions. Our research teams at Wayne State University and the University of Pennsylvania are collaborating to investigate this hypothesis. Supernatants (Sup) obtained from cultures of circulating B cells from MS patients are cytotoxic to both OL and neurons in vitro; those from normal controls (NC) show little or no toxicity (Lisak et al. 2012, 2017). Killing is independent of complement, and does not correlate with Sup levels of IgG, IgM or any single or combination of a large number of soluble cytokines and other proteins. Death of OL and neurons involves apoptosis and is caused by one or more factors in the >300 kDa fraction of MS B cell Sup (Lisak et al. 2017). We recently discovered that the OL toxicity is found in exosome-enriched (Ex-En) extracellular vesicles (EVs) released by the B cells from patients with MS (Benjamins et al. 2019). We will identify candidate factor/s using proteomics, RNASeq and lipidomics. Blocking the production or action of the factor/s could decrease OL and neuronal damage in cortical MS lesions and thus prevent progressive disease and enhance capacity for remyelination and axonal protection. Anticipated new outcomes include (1) comprehensive molecular profiling and characterization of Ex-En EVs released by MS B cells, which has not been done before, and (2) identification of factors in or on the Ex-En EVs that could mediate the damage induced by B cells in cortical lesions in MS. The integrated multiple -omics approaches we propose will generate rich datasets that will provide insight into the fundamental biology of B cell EVs as well as the differences in B cell EV phenotype and function in MS patients compared to NC. This project is significant because of its strong potential for identifying new targets and strategies to decrease damage and promote repair of the CNS in patients with MS. This B cell toxicity may be particularly relevant to non-relapsing progressive pathophysiology ? a major unmet therapeutic need. Our project is innovative because the function, components and roles of Ex-En EVs released by MS B cells have not been previously investigated.